Distance-Hi-C: Creating Photo Activated X-linkers To Define Nuclear Architecture

 描述（由申请人提供）：DNA的核组织是复杂的，由多层组成。在最低分辨率下，染色体的大部分被包装在区域中，在更高水平上，染色体区域被组织在拓扑相关的结构域中，这些结构域为与启动子和远端调控元件（增强子）结合的转录因子（TF）的相互作用提供框架，最后，调控因子、RNA聚合酶II和染色质的结构相互作用导致受调控的基因表达。近年来，已经开发了许多称为染色质（构象）捕获（CC）的方法，并用于捕获构成基因组结构和潜在调控相互作用的动态和稳定的DNA接触。这些方法的一个主要限制是它们都依赖于单一的交联剂甲醛，它将DNA与蛋白质以及蛋白质与其他蛋白质交联。这使得对观察到的“DNA-DNA”接触的解释复杂化，并且它缺乏距离信息。在这里，我们提出了一个正交的策略，命名为距离-高-C（D-Hi-C），我们设计，测试和应用电池的光活化交联剂，旨在直接测量相互作用的网站之间的距离全基因组。这些二价交联剂由两个被接头分开的反应性基团组成。可通过光活化交联的DNA嵌入剂（即Pd-benzene）将用作反应基团。接头将具有不同的精确长度，并且在性质上是柔性的或刚性的，使得可以推断交联的基因座之间的三维距离。当在Hi-C方案中取代甲醛时，这种DNA特异性二价交联试剂产生空间限制，揭示了增强子-启动子相互作用，并可能允许以前所未有的精确度推断核基因组的3D排列。此外，D-Hi-C有望降低背景，并允许检查短距离和中等距离的相互作用，这是由目前的方法的高背景掩盖。除了掺入到连接产物中的生物素之外，向二价交联剂中加入基团如地高辛将能够更好地纯化，从而更深入地检查基因组相互作用。最后，在基因激活后的时间采样DNA距离提供了一种探索4D架构和设置关键限制的方法，以评估基因激活的机制。具体目的是1）合成一组二价交联剂并评价它们在体外交联DNA的能力; 2）在果蝇核模型中测试D-Hi-C交联剂相对于甲醛的作用; 3）将新的交联剂应用于第1层，ENCODE GM 12878和K562细胞系以测试它们在人细胞中的功效;和4）测试基因座特异性光交联，其将允许在基因活化的时间过程中更集中和彻底地检查基因座特异性与基因组的相互作用。这些方法的发展将广泛用于提供关键的见解基因调控机制，在正常的动物发育和体内平衡，并在疾病如癌症中出错。

 DESCRIPTION (provided by applicant): Nuclear organization of DNA is complex and consists of multiple layers. At the lowest resolution, large sections of chromosomes are packed in territories, at a higher level chromosomal regions are organized in topologically-associated domains that provide a framework for interactions of transcription factors (TFs) bound to promoters and distal regulatory elements (enhancers), and finally the structural interplay of regulators, RNA polymerase II, and chromatin then lead to regulated gene expression. In recent years, a number of methods called chromatin (conformation) capture (CC) have been developed and used to capture dynamic and stable DNA contacts that constitute genome architecture and potential regulatory interactions. A major limitation of these methods is that they all depend on a single crosslinker, formaldehyde, which crosslinks DNA to proteins as well as proteins to other proteins. This complicates the interpretation of the observed 'DNA-DNA' contacts and it lacks distance information. Here we propose an orthogonal strategy, named Distance-Hi-C (D-Hi-C), where we design, test, and apply a battery of photo-activated crosslinkers, designed to directly measure distances between interacting sites genome-wide. These bivalent crosslinkers consist of two reactive groups separated by a linker. DNA intercalaters that can be crosslinked by photo-activation (i.e. Psoralen) will be used as the reactive groups. Linkers will be of varying precise lengths, and either flexible or rigid in nature so that the 3-dimensional distance between crosslinked loci can be inferred. Such DNA-specific bivalent crosslinking reagents, when substituted for formaldehyde in Hi-C protocol, produces space constraints revealing enhancer- promoter interactions and potentially allowing the inference of the 3D arrangement of the nuclear genome with unprecedented precision. Moreover, D-Hi-C is expected to lower backgrounds and allow examination of short and moderate range interactions, which are obscured by high backgrounds of current methods. Addition of groups such as digoxigenin to the bivalent crosslinkers, in addition to biotin incorporated to the ligation products, will enable better purification and thus deeper examination of genomic interactions. Finally, sampling DNA distances in time following gene activation provides a means of exploring the 4D architecture and setting critical limits in evaluating mechanisms of gene activation. The specific aims are to 1) synthesize a battery of bivalent crosslinkers and evaluate their ability to crosslink DNA in vitro; 2) test D-Hi-C crosslinkers relative to formaldehyde in the Drosophila nuclei model; 3) apply new crosslinkers to tier 1, ENCODE GM12878 and K562 cell lines to test their efficacy in human cells; and 4) test locus-specific photo-crosslinking that will allow a more focused and thorough examination of locus-specific interactions with the genome in a time course of gene activation. The development of these methodologies will be broadly useful in providing critical insights into gene regulatory mechanisms that are operative in normal animal development and homeostasis and that go awry in diseases like cancer.